Molecular diagnosis of familial adenomatous polyposis

ABSTRACT

A two-pronged method for diagnosis of genetic diseases can detect mutations in about 87% of familial adenomatous polyposis (FAP) patients. One part of the diagnostic method employs in vitro protein synthesis from surrogate genes created by amplifying either cDNA or genomic DNA. The second part of the diagnostic method employs an allele-specific expression assay which distinguishes the amount of mRNA expressed in vivo from each of a patient&#39;s two alleles. These approaches are readily applicable to the diagnosis of other genetic diseases.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grantsCA-35494, CA-57345, and CA44688, awarded by the National Institutes ofHealth.

TECHNICAL FIELD OF THE INVENTION

The invention relates to the area of medical diagnostics. Moreparticularly it relates to the area of diagnostic genetics, where thepresence of mutations in certain genes presage particular diseasestates.

BACKGROUND OF THE INVENTION

Familial Adenomatous Polyposis (FAP) is an autosomal,dominantly-inherited syndrome characterized by the progressivedevelopment of hundreds of adenomatous colorectal polyps, some of whichwill inevitably progress to cancer. Although the clinical manifestationsof this syndrome and its variants (e.g. Gardner's, Turcot's syndromes)have been known for many years (Bussey, H. J. R., et al. (1990) FamilialAdenomatous Polyposis, 1-7), diagnosis still relies largely on theobservation of numerous colorectal polyps developing during the secondor third decade of life. Today, with FAP afflicting nearly one in eightthousand people (Alm, T. et al. (1973) Clin. Gastroenterol, 2:557-602)and twice that many at risk, there are over 50,000 individuals in theU.S. alone whose families could potentially benefit from genetictesting.

The first step towards genetic testing was achieved when inheritance ofFAP was linked to a small region of chromosome 5(5q21) (Herrera, L., etal. (1986) American Journal of Medical Genetics 25:473-476; Bodmer, W.F., et al. (1987) Nature 328:614-616; Leppert, M., et al. (1987)238:1411-1413). This set the groundwork for linkage studies using nearbypolymorphic DNA markers. Although linkage analysis can be useful in somesituations, it can benefit only a minority of FAP kindreds in practice(Petersen, G. M., et al. (1991) Gastroenterology 100:1658-1664;Cachon-Gonzalez, M. B., et al. (1991) Journal of Medical Genetics28:681-685; Spirio, L., et al. (1993) Am. J. Hum. Genet. 52:286-296).Direct genetic testing became feasible when the APC gene on chromosome5q21 was found to be mutated in the germline of FAP patients (Groden,J., et al. (1991) Cell 66:589-600; Joslyn, G., et al. (1991) Cell66:601-613; Kinzler, K. W., et al. (1991) Science 253:661-665; Nishisho,I., et al. (1991) Science 253:665-669). Interestingly, the APC gene isalso mutated frequently and early during sporadic colorectaltumorigenesis (Nishisho, I., et al. (1991) Science 253:665-669; Powell,S. M., et al. (1991) Nature 359:235-237; Miyoshi, Y., et al. (1992)Human Molecular Genetics 1:229-233).

Analyses of the entire coding region of APC gene have alloweddemonstration of inactivating mutations in 30 to 60% of FAP patientsdepending on the screening method applied (Miyoshi, Y., et al. (1992)Proc. Natl. Acad. Sci. USA 89:4452-4456; Groden, J., et al. (1993) Am.J. Hum. Genet. 52:263-272; Nagase, H., et al. (1993) in press). Theseanalyses were complicated by the varied nature of mutations which weredistributed over a large portion of the APC gene, encompassing over8,500 bp of open reading frame. Moreover, these mutations were mostlysingle base pair changes, small insertions or deletions. Present methodsfor identifying such mutations are often insensitive and always laborintensive.

There is a clear need in the art for a rapid and sensitive method fordetection of APC mutations.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method for detectingmutations in an APC gene.

It is another object of the invention to provide a pair of primers foramplifying a segment of the APC gene coding sequence to form templatesuseful for in vitro transcription and translation.

It is still another object of the invention to provide a set of primersfor amplifying all segments of the APC gene coding sequence to formtemplates useful for in vitro transcription and translation.

It is yet another object of the invention to provide a method fordetecting mutations in any gene.

These and other objects of the invention are provided by one or more ofthe embodiments described below. In one embodiment of the invention amethod is provided for detecting mutations in the APC gene. The method(termed herein an in vitro synthesis or IVS assay) comprises the stepsof: forming APC templates by amplifying some or all portions of APC genecoding sequences in a DNA sample of a human; making a polypeptideproduct from said APC templates in in vitro transcription andtranslation reactions; analyzing said polypeptide products to determinethe size of said polypeptide products, a truncated polypeptide productindicating a mutation in an APC gene in said DNA sample.

If no mutations leading to truncated polypeptides are found by the invitro synthesized protein method described above, further steps can betaken to determine if a cis-acting mutation is present which reduces theamount of APC mRNA expressed. The method (termed herein as anallele-specific expression or ASE assay) comprises the further steps of:determining whether the patient is heterozygous for a polymorphism inthe APC gene, a patient who is heterozygous having a first and a secondpolymorphic allele of an APC gene; and determining the relative amountof mRNA transcribed from each of said two polymorphic alleles in a DNAsample of a heterozygous patient, a ratio of mRNA transcribed from saidfirst allele to mRNA transcribed from said second allele which isgreater than 1.2 or less than 0.8 indicating a mutation in one of saidalleles in the DNA. This method (ASE assay) can also be applied directlyto samples without first determining an absence of truncated polypeptideproducts in an IVS assay.

Also provided by the present invention are a pair of primers foramplifying all or a segment of APC gene coding sequence to formtemplates useful for in vitro transcription and translation. One of thepair of primers comprises a transcriptional promotor and a translationalinitiation site. Each of the primers comprises at least about twentycontiguous nucleotides which are complementary to opposite strands ofsaid gene coding sequence.

In another embodiment of the invention a set of primers is provided foramplifying all segments of APC gene coding sequence to form templatesuseful for in vitro transcription and translation. One primer in eachpair of the set of primers comprises a transcriptional promotor and atranslational initiation site. Each of said primers comprises at leastabout twenty contiguous nucleotides which are complementary to oppositestrands of said gene coding sequence.

In still another embodiment of the invention a method is provided fordetecting mutations in a gene. The method (an IVS assay) comprises thesteps of: forming templates by amplifying some or all portions of thecoding sequences of the gene in a DNA sample of a patient suspected ofbeing affected by, or a carrier of, a disease caused by mutation in thegene; making a polypeptide product from said templates in in vitrotranscription and translation reactions; analyzing said polypeptideproducts to determine the size of said polypeptide products, a truncatedpolypeptide product indicating a mutation in the gene in said DNAsample.

If no mutations leading to truncated polypeptides are found by the IVSassay described above, further steps can be taken to determine if acis-acting mutation is present which reduces the amount of mRNAexpressed from the gene. The further steps (an ASE assay) comprise:determining whether the human is heterozygous for a polymorphism in thegene, a human who is heterozygous having a first and a secondpolymorphic allele of the gene; determining the relative amount of mRNAtranscribed from each of said two polymorphic alleles in a DNA sample ofa heterozygous patient, a ratio of mRNA transcribed from said firstallele to mRNA transcribed from said second allele which is greater than1.2 or less than 0.8 indicating a mutation in one of said alleles in theDNA sample. The ASE assay can be performed even if an IVS assay has notfirst been performed to reveal truncated polypeptide products.

The present invention thus provides the art with a practical andsensitive screening method for detecting mutations in large genes, suchas APC, which are otherwise difficult to determine absent extremelylabor-intensive efforts. The invention allows routine preclinicaltesting of at-risk individuals, as well as genetic confirmation of denovo cases. Such methods will improve management of patients andpotentiate the development of effective non-invasive preventativemeasures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show schematics of the in vitro synthesis (IVS) andallele-specific expression (ASE) assays.

FIG. 1A shows a schematic illustrating the principles of the IVS assay.The APC gene is divided into five overlapping segments encompassing theentire coding region of the APC gene. These regions are amplified usingspecially designed PCR primers which place the necessary transcriptionaland translational regulatory sequences at the 5' end of the PCR product.Radiolabeled polypeptide is synthesized in vitro from these surrogategenes in a simple one-step coupled transcription-translation reaction(illustrated as two steps). Truncating mutations can then be identifiedas smaller polypeptide products after gel electrophoresis andautoradiography. The stop codon represents a typical truncating APCmutation, for example, a single base-pair change that creates apremature translation termination codon.

FIG. 1B is a schematic which shows an allele-specific-expression assay.Every normal cell has two copies of the APC gene that are identicalexcept for occasional polymorphisms of a single base pair (cytosine C!or thymidine T! in this example). Normally, both alleles of the APC geneare equally represented in the RNA fraction of the cell. However, somecases of familial adenomatous polyposis are caused by mutations thatlead to reduced levels of normal APC transcript from one allele. Thisresults in an imbalance in the representation of the transcripts fromthe two alleles. This altered allele ratio in RNA can be detected withthe allele-specific-expression assay (which is described below thedotted line). First, RNA is isolated from peripheral blood mononuclearcells. APC transcripts are converted to complementary DNA and amplifiedby reverse transcriptase PCR. The PCR products are then annealed with acommon 9-bp oligomer and two different-sized allele-specific oligomers(8 and 10 bp). After ligation, these oligomers will yield 17-bp and19-bp products corresponding to alleles A and B that can bedistinguished by gel electrophoresis. The box shows the expected resultsfrom a normal subject and from a patient with familial adenomatouspolyposis who has a mutation that leads to the reduced expression of thenormal transcripts of allele A.

FIGS. 2A and 2B show IVS polypeptide analysis for detection of knowntruncating APC mutations.

FIG. 2A shows representative IVS polypeptide samples of sporadiccolorectal tumors (T1-T8), known to have truncating mutations fromsequence analysis, run on SDS-PAGE. Each demonstrates the expectedtruncated APC polypeptide in segment 3. A significant amount of normal,full sized APC protein (Indicated at left by N) is noted from theremaining normal alleles. A normal tissue sample (Indicated by an N attop) is also shown.

FIG. 2B graphically depicts the predicted size of truncated APCpolypeptides in each of the tumors, based on sequence analysis of theAPC gene.

FIGS. 3A, 3B and 3C show IVS protein analysis for detection of APCmutations in FAP patients.

Representative samples of FAP subjects demonstrating truncated APCpolypeptides (indicated by a + at bottom and by an asterisk next to thebands) in segment 1 (S1), segment 2 (S2), and segment 3 (S3). Thenormal-sized, full-length APC protein (indicated at left by N) from theremaining unaltered APC allele is noted as well as background bands(also labelled N) which were noted in all lanes. The background bandsprobably result from internal initiation of protein translation. FAPsubjects without a demonstrable truncated APC protein in the segmentsanalyzed are also shown (Indicated by a - at the bottom).

FIGS. 4A and 4B show allele mixing analysis of the ASE assay.

Defined amounts of RNA, each homozygous for one allele (A or B) at theexon 11 polymorphic site, were used as templates for amplification andsubsequent ligation reactions. The input ratio for each allele is shownbelow FIG. 4A. A linear correlation between input and the observedresult in the ASE assay is apparent from FIG. 4A and the graph (FIG.4B). The correlation coefficient, r, was 0.997.

FIG. 5 shows detection of altered APC transcripts by the ASE assay.

Significantly reduced amounts of expression from an APC allele weredetected in three FAP patients using ASE analysis with the exon 11polymorphism. The "Genomic" bar indicates the average allele ratioderived from assays of 28 genomic DNA samples from 21 differentindividuals. The "Normal" bar indicates the average allele ratioobserved in eleven RNA samples from four different individuals. The "FAP#11", "FAP #38", and "FAP #62" bars represent analysis of RNA samplesfrom three different FAP patients. The ratio for each patient wasderived from four assays. Means and standard deviations are indicated bythe box and overlying brackets, respectively.

FIG. 6 shows the segments of APC used for analysis, as mapped to the APCexons.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is a discovery of the present invention that inactivating mutationsin large genes can be directly identified by a practical moleculargenetic approach. The approach disclosed here has several advantagesover currently available genetic methods. Although useful, geneticlinkage analysis cannot be applied in individuals where kindreds aresmall, requisite affected kindred members are not available, orpolymorphic markers are uninformative. Furthermore, linkage analysiscannot be applied when a de novo mutation is suspected. Indeed, de novocases account for approximately one third of FAP cases (Bulow, S. (1986)Dis. Colon Rectum 29:102-7) and accounted for 14 of the cases in thisstudy, 12 of which displayed APC mutations by our analysis.Additionally, because linkage analysis is indirect, some degree ofuncertainty always remains.

Several studies have described the direct detection of APC mutations inFAP patients with detection rates ranging from 10 to 60% depending onthe method applied (Groden, J., et al. (1991) Cell 66:589-600; Miyoshi,Y., et al. (1992) Proc. Natl. Acad. Sci. USA 89:4452-4456; Groden, J.,et al. (1993) Am. J. Hum. Genet. 52:263-272; Nagase, H., et al. (1993)Human Mutation (in press); Fodde, R., et al. (1992) Genomics13:1162-1168; Cottrell, S., et al. (1992) Lancet 340:626-630; Olschwang,S., et al. (1993) Am. J. Hum. Genet. 52:273-279; Varesco, L., et al.(1993) Am. J. Hum. Genet. 52:280-285). The screening methods employedwere generally very labor intensive and could miss a substantialfraction of subtle single base pair changes. In many cases the entireAPC gene was not examined, presumably due to practical considerationsrelated to its large size. Western blot analysis has been used to detecttruncated APC polypeptides and can be useful in some cases (Smith, K.J., et al. (1993) Proc. Natl. Acad. Sci. USA (in press)). However, manyof the truncated APC polypeptides in FAP are unstable in vivo,precluding their demonstration by Western blot assays (Smith, K. J., etal. (1993) Proc. Natl. Acad. Sci. USA (in press)). For example,truncated APC polypeptides could not be identified in three of seven FAPcases examined by Western blot analysis, but mutations in all sevencases were easily detectable through the present approach.

Analysis of polypeptide synthesized from surrogate APC genes can rapidlyidentify mutations which result in truncated APC polypeptide, whethercaused by splicing mutations, point mutations, or frameshifts. Likewise,ASE analysis identifies cis-acting mutations which alter APC expression,observed as an imbalance in the representation of alleles at the RNAtranscript level. A variety of events including promotor mutations,splicing mutations, mutations altering transcript stability, and evenimprinting abnormalities have the potential to be detected by thisanalysis. These molecular approaches are applicable to theidentification of mutations that lead to shortened polypeptides orreduced expression in any gene, but are especially useful in analysis oflarge genes where conventional methods are too labor intensive. Forexample, most mutations in the recently isolated genes responsible forneurofibromatosis type 2 (Trofatter, J. A., et al. (1993) Cell 72:791;Rouleau, G. A., et al. (1993) Nature 363:515) and von Hippel-Lindaudisease (Latif, F., et al. (1993) Science 260:1317) can be detected byour assays. Similarly most mutations in cystic fibrosis can be detectedby these assays.

The detection of APC alterations in 87% of FAP patients illustrates theusefulness of this approach as a genetic test for FAP. Moreover, theability to use the assay for prenatal diagnosis could be important toFAP patients planning a family. In providing a test for routinepresymptomatic testing, our assays provide significant practicalbenefits for FAP kindreds. Registry records for the 54 positive patientsshow that there are at least 280 relatives at risk for FAP who can nowbe tested. One hundred sixty-six of these individuals are under the ageof 20 and stand to gain the most from this analysis. Individuals whotest positive can at least be spared the anxiety associated with diseaseuncertainty and can benefit from improved management. Early diagnosisshould ensure that appropriate preventative measures are taken wellbefore the inevitable development of colorectal cancers. The importanceof preclinical testing is further heightened by recent studies showingpromise in the pharmacologic treatment of polyposis (Waddell, W. R., etal. (1983) Journal of Surgical Oncology 24:83-87; Waddell, W. R., et al.(1989) American Journal of Surgery 157:175-179; Rigau, J., et al. (1991)Annals of Internal Medicine 115:952-954; Labayle, D., et al. (1991)Gastroenterology 101:635-639; Giardiello, F. M., et al. (1993) NewEngland Journal of Medicine 328:1313). Such treatment of individuals whohave inherited a mutated APC gene should be more efficacious if providedas a preventative measure before polyps or other clinical manifestationsappear.

According to one aspect of the present invention, specific gene codingsequences are amplified. This can be done with any technique known inthe art, including polymerase chain reaction (Saiki et al., Science329:487-491, 1988), and ligase chain reaction (Wu et al. Genomics,4:560-569,1989). Gene coding sequences can be amplified using reversetranscription of mRNA to form a cDNA template, or by using portions ofgenomic sequences which are free of intervening sequences.

DNA samples for testing may be obtained from particularly affectedtissues, such as colorectal tumors, or from other tissues, such asperipheral blood, chorionic villi, and blastomeres of pre-implantationembryos. DNA samples are obtained from persons who are suspected ofbeing affected by, or of being a carrier of, a disease which is causedby mutation of the gene to be tested. For example, a DNA sample would beobtained from a person at risk of having familial adenomatous polyposisto test for mutations in the APC gene.

Templates which are formed by the amplification of portions of thedesired gene preferably will contain a transcriptional promotor and atranslational initiation site. These are conveniently attached to thetemplate during amplification by incorporating such sequences on theamplification primer which is complementary to the antisense strand ofthe gene. Alternatively, these can be attached by a separate ligationreaction. Upon transcription and translation of the amplified DNA(templates), a polypeptide is produced which contains all or a portionof the amino acid sequence of the gene being tested. In vitrotranscription and translation (IVS) can be accomplished in anyconvenient system known in the art, including rabbit reticulocytelysates and wheat germ lysates.

Polypeptides which are produced in the IVS are typically labeled with aradioactive substance or a fluorescent substance. For example, ³⁵S-methionine can be used as a substrate during the IVS to produceradiolabeled products which can be separated on an SDS-polyacrylamidegel and autoradiographed. Other means for size separation or detectionof polypeptide products as are known in the art may also be used.Mutations which are detectable by the IVS technique are those which leadto truncated polypeptides, which include nonsense and frameshiftmutations.

If no truncated polypeptide products are detected by the IVS assay, onecan test for mutations which cause a reduced amount of expression.According to the method of the present invention, this can be done ifthe patient is determined to be heterozygous for a polymorphism in thegene being investigated. Certain polymorphisms are already known in theart and can be used for this purpose, but others may be used as found.Coding change "silent" polymorphisms (i.e., those without apparenteffect on gene expression or protein function) are known in APC codons486, 545, 1493, 1756, 1960, 1678, and 2568. See Powell et al, Nature359:235-237, 1992, the disclosure of which is expressly incorporatedherein. The presence of such polymorphisms in the patient can beassessed, inter alia, by using the ligase-mediated gene detectiontechnique, generally as taught by Ladegren et al., Science241:1077-1080, 1988, the disclosure of which is expressly incorporatedherein. The template for such a ligase-mediated reaction can be genomicDNA.

If the patient is found to be heterozygous for one or more of thepolymorphisms, then the patient's mRNA can be used as a template in asecond ligase-mediated assay. The relative amount of mRNA transcribedfrom each of the two polymorphic alleles in the DNA sample of thepatient can be determined. If the ratio is more than 1.2 or less than0.8, then a mutation is indicated in one of the two polymorphic alleleswhich affects the expression of the gene. Such mutations include suchcis acting mutations as promotor mutations and splice site mutations.

An allele-specific ligation assay anneals two oligonucleotidesimmediately adjacent to each other on a complementary target DNAmolecule. Two versions of the adjacent oligonucleotide are joinedcovalently by the action of a DNA ligase, provided that the nucleotidesat the junction are correctly base-paired. One of the twooligonucleotides to be ligated is labeled. This oligonucleotide iscomplementary to DNA immediately adjacent to the polymorphism. Thesecond and a third oligonucleotides are not labeled, and each of thesecontains as its terminal nucleotide (adjacent to the firstoligonucleotide when annealed) one of the two polymorphic variantsrelevant to the patient under evaluation. The lengths of the twoversions of the second oligonucleotide are distinct from each other.Thus, when hybridized to the complementary target cDNA molecules andligated, the products of the ligation reaction will be distinguishableon the basis of size. The amount of the ligation products will beproportional to the amount of mRNA which was expressed in the samplefrom each allele. The ligation products can be separated on sequencinggels, for example, and the amount of each product determined with aphosphor imager. If the ratio of mRNA made from one allele to mRNA madefrom the other is outside of the range of 1+/-0.2, a mutation isindicated.

Large genes are particularly suitable for the present analysis, althoughit can be applied to any gene. Diseases caused by truncating-typemutations are preferred. These include, but are not limited to, cysticfibrosis, yon Hippel-Lindau disease, familial adenomatous polyposis,Hereditary Non-Polyposis Colon Cancer (HNPCC), and neurofibromatosistype 2.

Primers are provided for amplification of particular portions or entiregenes. These primers are paired, and can be used as individual pairs oras sets which will amplify a whole coding sequence. Each primer istypically ten or more bases in length. The sequence of the primer isprimarily complementary to one strand of the gene coding sequence,preferably about fifteen or twenty contiguous bases of the gene codingsequence. Each primer of a pair hybridizes and is complementary toopposite strands of the coding sequence. Desirably one primer of eachpair will incorporate the sequence of a promotor and translationalinitiation sequence. This primer is complementary to the antisensestrand of the gene to be amplified. Any promotor and initiation sequencecan be used, that are compatible with the IVS system to be used. Onepreferred promotor and initiation sequence is that of T7. Others as areknown in the art can be used.

EXAMPLES Example 1

This example demonstrates that the in vitro synthesized (IVS) proteinassay is a valid way to detect truncating mutations.

We analyzed 20 sporadic colorectal tumors which had previously beenshown to have truncating APC mutations by sequence analysis (Powell, S.M., et al. (1991) Nature 359:235-237). In each case, a specifictruncated polypeptide corresponding to the size of the predicted mutantproduct could easily be identified (examples in FIG. 2). The full lengthprotein product from the remaining normal allele was also noted in eachcase.

In vitro Synthesized (IVS) Protein Assay

For the purposes of PCR, the APC gene was divided into five overlappingsegments (#1 to 5) containing codons 1 to 804, 686 to 1217, 1099 to1693, 1555 to 2256, and 2131 to 2843 of APC, respectively (see FIG. 6).The primers used for PCR amplification were designed to introduce a T7promotor sequence for initiation of transcription by T7 RNA polymeraseas well as a consensus sequence for translation initiation (Kozak, M.(1987) Nucleic Acids Research 15:8125-8133). Segment 1 was isolated fromcDNA templates prepared by reverse transcribing mRNA. Segments 2 to 5were isolated directly from genomic DNA. For segment 1, randomly primedcDNA template was prepared by incubating 5 μg of total RNA, 1 μg ofrandom hexamer, 1 μl of amplification enhancer (0.5 units/μL; USB) and300 units of superscript II reverse transcriptase (BRL) in 20 μl ofreaction buffer for one hour at 37 degrees. As a control forcontamination, a mock reverse transcriptase reaction includingeverything except the enzyme was performed in parallel and used as a PCRtemplate. Two-stage, nested PCR was performed using the USB Bind-Aidamplification kit according to manufacturer's instructions with thefollowing changes. For the first stage, 4 μl of cDNA template, 35 ng ofeach outside primer (see below), and 2.5 units of Taq polymerase (Cetus)were used in a 20 μl PCR reaction for 10 cycles (95° C.×30', 62.5°C.×2', 70° C.×3') in a total volume of 50 μl.

For segments 2-5, 100 ng of genomic DNA, 350 ng each of the appropriateprimers (see below), and 5.0 units of Taq polymerase were used in a 50μl Bind-Aid amplification kit (USB) reaction. Amplification wasperformed for 35 cycles of 30" denaturation (95° C.), 90" annealing(segment 2, 60° C.; segment 3, 65° C.; segment 4, 62.5° C., segment 5,60° C. and 90" extension (70° C.). All PCR reactions included a 5 minuteextension period.

PCR products were used directly (without purification) as templates in25 μl coupled transcription-translation reactions (Promega) containing40 μCi of S³⁵ -Methionine translabel (ICN) for one hour at 30° C.Samples were diluted in sample buffer, boiled for 5', and one tenthanalyzed on 10%-20% gradient SDS-poly acrylamide gel. Polypeptides werevisualized by fluorography after impregnating the gel with ENHANCE(NEN). The sequence of amplification primers were as follows withT7-trans! (SEQ ID NO:23 representing the sequence displayed in FIG. 6:

Segment 1

    __________________________________________________________________________    External: 5'-CAAGGGTAGCCAAGGATGGC-3' SEQ ID NO: 11 and                        5'-TTGCTAGACCAATTCCGCG-3' SEQ ID NO: 12                                       Internal: 5'- T7-trans!-CTGCAGCTTCATATGATC-3' (SEQ ID NO: 1) and              5'-CTGACCTATTATCATCATGTCG-3' (SEQ ID NO: 2)                                   Segment 2: 5'- T7-trans!-ATGCATGTGGAACTTTGTGG-3' (SEQ ID NO: 3) and           5'-GAGGATCCATTAGATGAAGGTGTGGACG-3' (SEQ ID NO: 4)                             Segment 3: 5'- T7-trans!-TTTCTCCATACAGGTCACGG-3' (SEQ ID NO: 5) and           5'-GGAGGATCCTGTAGGAATGGTATCTCG-3' (SEQ ID NO: 6)                              Segment 4: 5'- T7-trans!-AAAACCAAGAGAAAGAGGCAG-3' (SEQ ID NO: 7) and          5'-TTCACTAGGGCTTTTGGAGGC-3' (SEQ ID NO: 8)                                    Segment 5: 5'- T7-trans!-GTTTATCTAGACAAGCTTCG-3' (SEQ ID NO: 9) and           5'-GGAGTGGATCCCAAAATAAGACC-3' (SEQ ID NO: 10)                                 __________________________________________________________________________

Example 2

This example demonstrates that the IVS protein assay can be used todiagnose FAP from peripheral blood samples.

Analysis of the entire coding region of APC with the IVS protein assayidentified truncating mutations in 51 (82%) patients (examples in FIG.3). The 51 mutations were distributed over the first four segments with29, 10, 11 and 1 being in segments 1, 2, 3 and 4, respectively.

Study Group

The most recent 62 unrelated patients enrolled in the Johns Hopkins FAPregistry from whom blood samples could be obtained were selected foranalysis in an unbiased manner. All of these individuals were confirmedto have classic adenomatous polyposis as defined by the presence ofgreater than 100 colorectal polyps at time of diagnostic endoscopy,radiologic study, or examination of surgically resected colons. Theadenomatous nature of the polyps was documented by histopathologicalanalysis.

Blood samples were also collected from nine healthy individuals, age22-43, as normal controls and from seven relatives of three FAPpatients. Informed consent in accord with institutional policy wasobtained from each subject prior to drawing blood samples.

Preparation of Templates

Whole blood samples were obtained from 45 FAP patients, and consisted of20 cc EDTA anticoagulated whole blood collected and stored overnight atambient temperature. Genomic DNA was prepared by Chelex extraction of 30μl of whole blood as described previously (Walsh, P. S., et al. (1991)Biotechniques 10:507-513). RNA was isolated by the acid guanidiumisothiocyanate-phenol-chloroform extraction method (Chomczynski, P., etal. (1987) Analytical Biochemistry 162:156-159) from peripheralmononuclear cells prepared from 10 cc of whole blood using aHistopaque-1077 (Sigma) gradient. Lymphoblastoid cell lines wereobtained from 17 FAP patients by Epstein Barr virus immortalization oftheir lymphocytes. RNA and DNA were extracted from such cells asdescribed (Chomczynski, P., et al. (1987) Analytical Biochemistry162:156-159; Goelz, S. E., et al. (1985) Biochemical and BiophysicalResearch Communications 130:118-126).

Example 3

This example demonstrates the accuracy of the Allele-specific Expression(ASE) assay.

Considering that some cases of FAP might result from promotor orsplicing mutations that lead to reduced levels of normal APCtranscripts, we additionally addressed ways to easily identify suchmutations. Because these cis acting mutations could at most result in a50% decreased level of total transcript, we needed to devise a sensitiveassay for expression of each of the two alleles individually. Therefore,we took advantage of single base pair polymorphisms to create anAllele-specific Expression (ASE) assay to detect this type of alteration(FIG. 1B). The accuracy of this assay was demonstrated in an allelemixing experiment.

A controlled amount of RNA from two patients each homozygous fordifferent alleles at the polymorphic site in exon 11 were mixed togetherin defined ratios, amplified, and analyzed by the ASE assay. Relativeallele amounts determined by ASE analysis of these samples proved to belinear and quantitative with respect to the predicted ratios (r=0.997,FIG. 4).

Allele-specific Expression (ASE) Assay

The two site polymorphisms used in this assay were silent single basepair changes, one in exon 11 (codon 486, TAC/TAT) and one in exon 13(codon 545, GCA/GCG) (Powell, S. M., et al. (1991) Nature 359:235-237).Homozygous cases were first sought by analyzing amplified APC exons 11and 13 from genomic DNA.

Amplification of exon 11 and 13 was carried out using approximately 100ng of genomic DNA, 350 ng each primer, and 2.5 units of Taq polymerasewas used in a 50 μl Bind-Aid amplification kit (USB) reaction was cycled35 times 30' denaturation (95° C.), 1' annealing (exon 11, 52.5° C.;exon 13, 55° C.) and 1' extension (70° C.)! All reactions included a 5'extension period at the end. The sequence of primers used foramplification were as follows: (exon 11, 5'-TAGATGATTGTCTTTTTCCTC-3' SEQID NO:13, 5'-TCATACCTGAGCTATCTTAAG-3' SEQ ID NO:14; exon 13,5'-ACATGAAATTCATATTATAGTAC-3' SEQ ID NO:15, 5'-CTATTCTTACTGCTAGCATTA-3'.The genomic PCR product was added to a modified allele-specific ligationassay (Landgren, et al. supra, which is incorporated herein byreference). A common 9 base pair ³² P-labeled oligomer and two differentsized allele-specific oligomers (8 and 10 base pairs) were used in theligation assay.

Specifically, one μl genomic PCR product was added to 2 ng of a common³² P-5' labeled oligomer (exon 11, 5'-GGGCTTACT-3' SEQ ID NO:17; exon13, 5'-AGTGTTTTGA-3') SEQ ID NO:18) and 2 ng of allele-specificoligomers (exon 11, 5'-TGAAATGTAC-3' SEQ ID NO:19 and 5'-AAATGTAT-3';SEQ ID NO:20; exon 13, 5'-GGTTATTGCA-3' SEQ ID NO:21 and 5'-TTATTGCG-3'SEQ ID NO:22) in a modified ligase-mediated reaction. Ligation productswere separated through polyacrylamide/8M urea sequencing gels and theabundance of each allele was determined by the relative amount ofallele-specific ligation product (17 bp for allele A, 19 bp for alleleB, see FIG. 4). Quantitation was achieved with a PhosphorImager™(Molecular Dynamics, Inc.). Segment 1 PCR products (derived from mRNA,as for the IVS protein assay) were then analyzed in the samequantitative ligation reaction to determine the relative abundance ofAPC transcripts expressed from each allele.

Example 4

This example demonstrates the use of the ASE assay to evaluate patientsand healthy controls with no detectable protein abnormalities in the IVSassay.

We used the ASE assay to evaluate the eleven FAP patients withoutdetectable APC protein abnormalities as well as nine healthy non-FAPcontrols. Seven patients and six normal individuals were heterozygousfor at least one of the two polymorphisms and could thereby be analyzed.The relative allele ratio was 1+/-0.15 for all the genomic DNA samples(FAP or normal individuals) and for the RNA from control individuals.Three of the FAP patients had significantly reduced expression from oneallele (FIG. 5). In each of these three cases, the ratio of alleleabundance from expressed RNA template was significantly different fromthat of normal individuals and from the ratios found using genomic DNAinstead of RNA as template (P<0.001, two tailed unpaired Student's ttest). At least one other affected family member from the kindreds ofthese three patients was also analyzed and found to display similarlyreduced expression of the same allele, demonstrating the expectedinheritance.

Combined, these two assays (IVS protein and ASE) successfully identifiedAPC mutations in 87% of the 62 different FAP kindreds tested.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 23                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGATCCTAATACGACTCACTATAGGGAGACCACCATGGCTGCAGCTTCATATGATC56                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTGACCTATTATCATCATGTCG22                                                      (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGATCCTAATACGACTCACTATAGGGAGACCACCATGGATGCATGTGGAACTTTGTGG58                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GAGGATCCATTAGATGAAGGTGTGGACG28                                                (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GGATCCTAATACGACTCACTATAGGGAGACCACCATGGTTTCTCCATACAGGTCACGG58                  (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGAGGATCCTGTAGGAATGGTATCTCG27                                                 (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GGATCCTAATACGACTCACTATAGGGAGACCACCATGGAAAACCAAGAGAAAGAGGCAG59                 (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TTCACTAGGGCTTTTGGAGGC21                                                       (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGATCCTAATACGACTCACTATAGGGAGACCACCATGGGTTTATCTAGACAAGCTTCG58                  (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GGAGTGGATCCCAAAATAAGACC23                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CAAGGGTAGCCAAGGATGGC20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TTGCTAGACCAATTCCGCG19                                                         (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TAGATGATTGTCTTTTTCCTC21                                                       (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TCATACCTGAGCTATCTTAAG21                                                       (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ACATGAAATTCATATTATAGTAC23                                                     (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CTATTCTTACTGCTAGCATTA21                                                       (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GGGCTTACT9                                                                    (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AGTGTTTTGA10                                                                  (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TGAAATGTAC10                                                                  (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AAATGTAT8                                                                     (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GGTTATTGCA10                                                                  (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      TTATTGCG8                                                                     (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GGATCCTAATACGACTCACTATAGGGAGACCACCATGG38                                      __________________________________________________________________________

We claim:
 1. A method for detecting mutations in an APC gene, comprisingthe steps of:forming APC templates by amplifying some or all portions ofAPC gene coding sequences in a DNA sample of a human; making polypeptideproducts from said APC templates in in vitro transcription andtranslation reactions; analyzing said polypeptide products to determinethe size of said polypeptide products, a truncated polypeptide productindicating a mutation in an APC gene in said DNA sample.
 2. The methodof claim 1 further comprising the steps of:determining whether the humanis heterozygous for a polymorphism in the APC gene, a human who isheterozygous having a first and a second polymorphic allele of an APCgene; determining the relative amount of mRNA transcribed from each ofsaid two polymorphic alleles in a DNA sample of a heterozygous human, aratio of mRNA transcribed from said first allele to mRNA transcribedfrom said second allele which is greater than 1.2 or less than 0.8indicating a mutation in one of said alleles in the DNA sample.
 3. Themethod of claim 1 wherein the DNA sample is from a colorectal tumor. 4.The method of claim 1 wherein the DNA sample is from peripheral blood.5. The method of claim 1 wherein the DNA sample is from prenatal orembryonic cells.
 6. The method of claim 1 wherein the step of formingtemplates employs primers which introduce a promotor sequence forinitiation of transcription and a sequence for translation initiation.7. The method of claim 1 wherein the step of forming templates employsprimers which introduce a T7 promotor sequence for initiation oftranscription by T7 RNA polymerase.
 8. The method of claim 1 wherein thestep of forming templates employs primers having the sequences shown inSEQ ID NOS:1-10.
 9. The method of claim 1 wherein the step of formingtemplates employs cDNA templates for amplifying exons 1-14 and genomicDNA for amplifying exon
 15. 10. The method of claim 1 wherein the stepof analyzing employs SDS-polyacrylamide gels to separate saidpolypeptide products on the basis of size.
 11. The method of claim 2wherein the polymorphism is a silent single base-pair change.
 12. Themethod of claim 11 wherein the polymorphism is present in a codonselected from the group consisting of: codons 486, 545, 1493, 1756,1960, 1678, and
 2568. 13. The method of claim 2 wherein the presence ofthe polymorphism is determined by means of an allele-specific ligationassay.
 14. The method of claim 2 wherein the relative amount of mRNAtranscribed from each polymorphic allele is determined by means of anallele-specific ligation assay.
 15. A method for detecting mutations inan APC gene, comprising the steps of:determining whether a human isheterozygous for a coding change silent polymorphism in the APC gene, ahuman who is heterozygous having a first and a second polymorphic alleleof an APC gene; determining the relative amount of mRNA transcribed fromeach of said two polymorphic alleles in a DNA sample of a heterozygoushuman, a ratio of mRNA transcribed from said first allele to mRNAtranscribed from said second allele which is greater than 1.2 or lessthan 0.8 indicating a mutation in one of said alleles in the DNA.
 16. Amethod for detecting refutations in an APC gene comprising the stepsof:determining whether a human is heterozygous for a silent singlebase-pair polymorphism in the APC gene, a human who is heterozygoushaving a first and a second polymorphic allele of an APC gene;determining the relative amount of mRNA transcribed from each of saidtwo polymorphic alleles in a DNA sample of a heterozygous human, a ratioof mRNA transcribed from said first allele to mRNA transcribed from saidsecond allele which is greater than 1.2 or than 0.8 indicating amutation in one of said alleles in the DNA.
 17. The method of claim 16wherein the polymorphism is present in a codon selected from the groupconsisting of: codons 486, 545, 1493, 1756, 1960, 1678, and
 2568. 18.The method of claim 15 wherein the presence of the polymorphism isdetermined by means of an allele-specific ligation assay.
 19. The methodof claim 15 wherein the relative amount of mRNA transcribed from eachpolymorphic allele is determined by means of an allele-specific ligationassay.
 20. A method for detecting mutations in a gene of a humansuspected of being a carrier of, or being affected by, a disease causedby mutation of the gene, the method comprising the steps of:formingtemplates by amplifying some or all portions of the coding sequences ofthe gene in a DNA sample of a human; making polypeptide products fromsaid templates in in vitro transcription and translation reactions;analyzing said polypeptide products to determine the size of saidpolypeptide products, a truncated polypeptide product indicating amutation in the gene in said DNA sample.
 21. The method of claim 20further comprising the steps of:determining whether the human isheterozygous for a polymorphism in the gene, a human who is heterozygoushaving a first and a second polymorphic allele of the gene; determiningthe relative amount of mRNA transcribed from each of said twopolymorphic alleles in a DNA sample of a heterozygous human, a ratio ofmRNA transcribed from said first allele to mRNA transcribed from saidsecond allele which is greater than 1.2 or less than 0.8 indicating amutation in one of said alleles in the DNA.
 22. A method of detectingmutations in a gene, comprising the steps of:determining whether a humanis heterozygous for a coding change silent polymorphism in the gene, ahuman who is heterozygous having a first and a second polymorphic alleleof the gene, determining the relative amount of mRNA transcribed fromeach of said two polymorphic alleles in a DNA sample of a heterozygoushuman, a ratio of mRNA transcribed from said first allele to mRNAtranscribed from said second allele which is greater than 1.2 or lessthan 0.8 indicating a mutation in one of said alleles in the DNA. 23.The method of claim 20, 21, or 22 wherein the gene is merlin, mutationof which is responsible for neurofibromatosis type
 2. 24. The method ofclaim 20, 21, or 22 wherein the gene is VHL, mutation of which isresponsible for von Hippel-Lindau disease.
 25. The method of claim 20,21, or 22 wherein the gene is CF, mutation of which is responsible forCystic Fibrosis.
 26. The method of claim 20, 21, or 22 wherein the geneis hMSH2, mutation of which is responsible for Hereditary Non-Polyposiscolon Cancer (HNPCC).
 27. The method of claim 21 or 22 wherein thepresence of the polymorphism is determined by means of anallele-specific ligation assay.
 28. The method of claim 21 or 22 whereinthe relative amount of mRNA transcribed from each polymorphic allele isdetermined by means of an allele-specific ligation assay.
 29. A methodfor detecting mutations in a gene, comprising the steps of:determiningwhether a human is heterozygous for a silent, single base-pairpolymorphism in the gene, a human who is heterozygous having a first andsecond polymorphic allele of the gene; determining the relative amountof mRNA transcribed from each of said two polymorphic alleles in a DNAsample of a heterozygous human, a ratio of mRNA transcribed from saidfirst allele to mRNA transcribed from said second allele which isgreater than 1.2 or less than 0.8 indicating a mutation in one of saidalleles in the DNA.